blood groups: genetics and physiology

Blood groups: genetics and physiologyJ.-P. CartronInstitut National de la Transfusion Sanguine, Paris, France

Numerous studies in biochemistry, genetics and molecular biology conducted overtwo decades have shown that blood group gene products expressed on red cells andvarious tissues exhibit a variety of potential functions, which can be schematicallyclassified into functional groups such as transporters and channels, receptors, adhe-sion molecules, enzymes or structural components, with a further level of complex-ity as a same molecule may exhibit multiple functions. As structures present on redcells, blood group and blood group-related molecules may contribute to the struc-ture and function of the red cell membrane and may help to clarify unresolved bio-logical processes (for instance, water and gas transport through biologicalmembranes). Alternatively, some of these molecules may simply be the witnesses ofa residual persistence of membrane components during erythroid cell differentia-tion with no significant function. Investigation of blood group and blood group-related molecules as structures present on epithelial or endothelial cells of tissuesand organs has revealed other features of their potential physiological role and ledto the discovery of some unexpected functions (for instance Fy-mediated transcy-tose). Increasing our knowledge on both aspects is of interest not only for transfu-sion medicine, but also for transplantation and for understanding the normalphysiology and physiopathology of several diseases. Current studies indicate thatblood group and blood group-related molecules cover large areas of human physi-ology from cerebral to renal and reproduction biology. As specific deficiencies ofthese molecules have only a minor or no detrimental effect in most instances, theyare either dispensable for cell function or redundant. In some instances, however, afunction becomes apparent under stress or in pathological conditions. How com-mon and rare blood group polymorphisms are maintained and may impact functionis largely unknown, although a few examples clearly point to selective pressureexerted by pathogenic micro-organisms. Despite significant progress, much remainsto be discovered to clearly delineate how blood group molecules, alone or as molec-ular complexes in erythroid and non-erythroid cells, may act in health and disease,to understand the underlying mechanisms, and, ultimately, how these findingsmight eventually be translated into clinical applications.

Key words: antigens, chemokine receptor, gas transport, gene targeting, nullphenotypes, red cells.

Introduction

In the past years, the discovery of blood groups has signifi-

cantly contributed to our knowledge of human genetics.

Currently, numerous studies in biochemistry, genetics and

molecular biology conducted over two decades have shown

that blood group gene products exhibit a variety of poten-

tial functions which can be schematically classified into

functional groups such as transporters and channels, recep-

tors, adhesion molecules, enzymes or structural compo-

nents, with a further level of complexity as a same

molecule may exhibit multiple functions [for reviews, see

1–7]. This is not surprising because although many blood

group molecules were ‘fished out’ by their antigenic

Correspondence: Jean-Pierre Cartron, Institut National de la TransfusionSanguine, 6, rue Alexandre Cabanel, 75739 Paris Cedex 15, FranceE-mail: [email protected]

ISBT Science Series (2010) 5, 27–45

STATE OF THE ART 3B-S03-02 ª 2010 The Author.Journal compilation ª 2010 International Society of Blood Transfusion

27

properties on the red-blood-cell (RBC) surface, most are

also expressed in non-erythroid tissues.

Currently, 30 blood group systems are identified, which

defined 270 antigens, but 38 other antigens in the collec-

tions and series have no molecular basis yet. The resolution

of the red cell membrane proteome will be useful to charac-

terize these molecules [8,9], and it is likely that some will

prove to be associated with new functions.

The biological function of blood group molecules, first

based mostly on structural criteria, can now be grasped in a

variety of ways, including experimental methods designed

with native cells, purified components or recombinant mol-

ecules in expression systems coupled to site-directed muta-

genesis. Physiological studies in humans are difficult or not

feasible, but studies of genetic variants in humans and in

animal models, or homologues in lower organisms, provide

another way to approach blood group function in vivo.

Recent advances from selected examples will be summa-

rized in this review.

Rh proteins and transport function

The Rh complex of the red cell membrane is an oligomeric

assembly (most likely a trimer) of two erythroid-specific

proteins, Rh (synonym Rh30) and RhAG (Rh-Associated

Glycoprotein, synonym Rh50), a glycoprotein strictly

required for cell surface expression of Rh proteins, to

which the accessory chains CD47, ICAM-4 and GPB are

associated by non-covalent bonds [10–13]. By interacting

with skeletal proteins via a physical linkage with Ankyrin-

R [14], the Rh complex contributes to regulate the shape,

deformability and mechanical properties of red cells

and disruption of this linkage may occur in RBCs from

patients with the Rhnull syndrome which is characterized

by the lack or severe decrease of the Rh complex [15]. A

further level of complexity is also revealed by an inter-

action with protein 4Æ2 and CD47 [16,17] and the relation-

ship with Band 3 to form a macrocomplex, presumably a

‘transport metabolon’, consisting of a physically associ-

ated complex of proteins involved in the sequential meta-

bolic pathway responsible for CO2 ⁄ O2 gas exchange in

lungs and tissues [18].

The Rh30 and RhAG proteins display significant

sequence similarities (�36%) and have a similar predicted

secondary structure with 12 transmembrane domains. They

are encoded by two genes, RH and RHAG, located on chro-

mosome 1p34-p36 and 6p12-p21, respectively. The RH

locus itself is composed of two homologous genes, RHD

and RHCE (96% homology), which are tandemly organized

in opposite orientations and encode the RhD (carrier of D

antigen) and RhCE (carriers of C ⁄ c and E ⁄ e antigens) pro-

teins, respectively. The RHAG locus defines a new blood

group system encoding the RhAG protein, carrier of two

high frequency antigens, Duclos and DSLK, and the low fre-

quency antigen Ola [19].

Blast similarity searches in databanks revealed that

Rh ⁄ RhAG proteins and Rh50-like genes in the nematode

Caenorhabditis elegans share sequence similarity (�20–

27%) with microbial ammonium transporters (Amt) and

methylammonium permeases (Mep) (Amt ⁄ Mep family),

which use ammonium as nitrogen source [20]. This was the

first clue for a putative function of Rh proteins and was fol-

lowed by the identification of many Rh homologues in dif-

ferent species of vertebrates, invertebrates and lower

primitive organisms.

Extensive phylogenetic studies [21–23] have shown that

in mammals, the RH gene family includes four subgroups

of genes, RH, RHAG, both erythroid specific, and RHBG

and RHCG on chromosomes 1q21 and 15q25, respectively,

which are present in epithelial cells of non-erythroid tissues

such as kidney, but also with a distinct distribution in liver,

skin, ovary (RhBG) and brain, pancreas, prostate, testis

(RhCG) [21,24]. RhBG and RhCG have a polarized expres-

sion in epithelial cells, with RhBG mainly sorted to the ba-

solateral domain and RhCG to the apical domain [24]. In

non-mammalian vertebrates (fish, frog, reptile), there is an

additional gene family called Rhp2 (p for primitive), which

might derive from a common ancestral gene that gives rise

to the RH gene family. In invertebrates and unicellular

microbes, the RH gene homologues belong to a ‘Rhp1 clus-

ter’ which did not split into subgroups as in mammals.

Rhp1 members exhibit genetic uniformity possibly reflect-

ing a conserved function, whereas the subgroups seen in

vertebrates might reflect some functional diversification

[21]. Thus, eukaryotic evolution was shaped by gene

expansion and contraction events from a single Rh gene in

unicellular organisms to six in teleost fish, four in mam-

mals (RH, RhAG, RHBG, RHCG) and finally five in humans

following tandem gene duplication of the RH locus into

RHD and RHCE genes. Additionally, changes in exon–

intron organization also occur from intronless genes in

bacteria to multiple exons in all metazoans. Positive and

negative selection forces probably acted to shape these pat-

terns, but their nature and the advantages they would have

confer to organisms are not known. Interestingly, the fast

coevolution of RH and RHAG observed in mammals is

synchrone with the transition towards enucleated bicon-

cave red cells that may favour gas exchange by increasing

surface area-to-volume ratio [22].

Schematically, members of the RH and AMT ⁄ MEP gene

family may coexist in all phyla except vertebrates. Only RH

genes are present in vertebrates but they are absent in

plants and fungi, whereas AMT genes have an opposite

organismal distribution. Of note, Rh homologues are virtu-

ally absent from bacteria and microbial eukarya, with the

notable exceptions of Nitrosomonas europaea (Ne) and

28 J.-P. Cartron

� 2010 The Author.Journal compilation � 2010 International Society of Blood Transfusion, ISBT Science Series (2010) 5, 27–45

Chlamydomonas reinhardtii (green alga), respectively, both

used as models to study the structure ⁄ function of Rh pro-

teins. Moreover, AMT genes have been lost in Nitrosomon-

as europaea. These studies point to the early origin of the

RH gene family and to a distant relationship between RH

and AMT genes family that suggest some functional diver-

sification from an ancestral role in ammonium transport to

a postulated implication in CO2 ⁄ O2 gas transport [21].

Structural modelling of Rh proteins

Three-dimensional structures for the ammonium transport-

ers of Escherichia coli and Archaeoglobus fulgidus [25–27],

as well as for the Rh homologue (NeRh50 protein) of the

ammonium oxidizing bacteria Nitrosomonas europaea

[28,29], have been solved recently, based on high resolution

X-ray structures. All models indicate that these proteins

exhibit a conserved packed trimeric structure, each subunit

with 11 transmembrane (TM) domains carrying a narrow

hydrophobic conduction pore connecting a periplasmic and

a cytoplasmic vestibule. These studies provided strong sup-

port to the view that these proteins act as gas channels for

NH3,

The permeation pore of bacterial Amt proteins (E. coli

and A. fulgidus) schematically includes four regions: (i) an

external vestibule with a NHþ4 -binding site where deproto-

nation could occur by cation-p interaction with conserved

aromatic residues (F103, F107, W148) and H-bonding with

S219; (ii) a ‘phenylalanine gate’ that obstructs pore entry

by two stacked phenylalanines (F107, F215) in parallel

orientation; transient structural fluctuations would be suf-

ficient for opening this gate, thus allowing small molecules

to pass (NH3, water); (iii) a narrow hydrophobic pore with

two strictly conserved almost coplanar H-bonded histidines

(H168 and H318) critical for substrate conductance [30], for

which energetic considerations suggest that only NH3 con-

duction is favoured; (iv) a cytoplasmic vestibule where NH3

is reprotonated.

The permeation pore of the NeRh50 protein from N. eu-

ropaea retains similarities with AmtB proteins, notably the

conserved twin-histidine residues (H146 and H300 in

NeRh50) in the conduction pore, but differs from bacterial

Amts in several ways: (i) the predicted NHþ4 -binding site in

the external vestibule is absent (W148 and F103 of Amt

replaced by L126 and I82, respectively, in NeRh50), which

should favour the conduction of NH3 by a pH-dependent

mechanism, and an altered orientation of F86 (equivalent

to Amt F107); (ii) a ‘phenylalanine gate’ with the F86 and

F194 residues positioned in perpendicular orientation

(entry larger than in AmtB), (iii) an additional a-helix-

folded cytoplasmic domain that might be important for the

binding of partners [29] or as an allosteric regulator of

function [31].

In silico homology models of human Rh glycoproteins

(RhAG, RhBG and RhCG) based on AmtB structure as tem-

plate [32,33] have been revised using the NeRh50 structural

model [4,34–36]. Like NeRh50, Rh glycoproteins lack the

NHþ4 -binding site present in AmtB (replaced by aliphatic

residues S160 and I116 in RhAG) and both the ‘phenylala-

nine gate’ (F120 and F225 in RhAG) and the twin-histidine

motif in the conduction pore (H175 and H334 in RhAG)

were conserved. The cytoplasmic vestibule of Rh glycopro-

teins should be investigated in more detail, because the

ammonium transport is considered as bidirectional, at least

when expressed in yeasts [37]. The resolution of the crystal

structure of human RhCG (pdb 3HD6) deposited in the

Brookhaven database by the group of R. Stroud, is in gen-

eral agreement with these findings, and both NeRh50 and

RhCG appear as the best structural models to study struc-

ture ⁄ function relationship of Rh glycoproteins. A schematic

representation of the human RhAG conduction pore tem-

plated on the RhCG model is shown in Fig. 1. Structural

modelling of the Rh proteins that carry the RhD and RhCcEe

antigens revealed that the critical residues depicted earlier

for the ammonium transport function are not conserved.

Notably, the ammonium-binding site in the external vesti-

bule is altered (I116 in RhAG replaced by R114), as well as

the ‘phenylalanine gate’ (replaced by M118 and F223 ⁄ V223

in RhD andRhCE) and the twin-histidines (replaced by Y173

and F332 in both RhD and RhCE proteins), which is consis-

tent with functional studies with natural mutants [38], and

murine Rh ⁄ Rhag gene knockout [39] showing that Rh

proteins do not transport ammonia.

In contrast to other members of the AmtB ⁄ Mep family,

topological models of mature Rh proteins predicted a 12-

TM structure. The extra-TM, called TM0 [32], was modelled

ab initio and this helix has been positioned in the periphery

of the oligomeric architecture in the external space remain-

ing between the subunits [33]. Moreover, it was predicted

that the interface in human mixed RhAG ⁄ Rh trimers

formed an inner ring stabilized by H-bonding through two

polar residues (Q ⁄ D) in M1 helices (RhAG Q55 ⁄ D56; Rh

Q52 ⁄ D53) [33]. Polar Q ⁄ D residues are strictly conserved in

human RhBG (Q65 ⁄ D66) and RhCG (Q64 ⁄ D65) proteins.

Whether the hydrophilic ‘inner ring’ in the mixed Rh ⁄ RhAG

trimer may form a pore permeable to a substrate, as

suggested for the ‘central pore’ between the subunits of the

tetrameric AQP1 water channel [40], is unknown. After

analysis of residues predicted to be located at the interface

between the subunits of the NeRh50 trimer, it was sug-

gested that mixed trimers (RhAG + Rh) are unlikely [28].

As discussed before [4,33], this prediction is in contradic-

tion with two main observations: (i) in Rhnull individuals,

when either RhAG or Rh proteins are lacking, the Rh

complex does not reach the RBC surface [15], which is

difficult to explain if these two molecules are completely

Blood groups 29


independent and not part of the same molecular assembly,

(ii) RhAG is strictly necessary for Rh membrane expression,

both in vivo (Rhnull) and in vitro [41]; although some RhAG

proteins may be expressed without Rh proteins, the reverse

is not true and to date there is no example in which human

Rh proteins can be expressed without the RhAG subunit. As

such RhAG plays the role of a chaperone for Rh. Thus, the

crystal structure of Rh-RhAG complexes, when resolved,

will be critical to characterize Rh oligomers, as they occur

on RBCs.

Ammonia transport

Experimental evidence first based on functional comple-

mentation in yeast Saccharomyces cerevisiae lacking the

three endogenous Mep ammonium transporter genes [37],

followed by expression studies in Xenopus laevis oocytes

and transfected eukaryotic cells, indicated that all Rh gly-

coproteins (RhAG, RhBG and RhCG) have the capacity to

mediate a facilitated bi-directional ammonium transport, as

discussed in an International Conference in Paris [42]. In

aqueous solutions, ammonium ions (NHþ4 ) and ammonia

(gas NH3) are in equilibrium (pKa = 9Æ25), and NHþ4 is by

far the major fraction (�99%) at a physiological pH of 7Æ35

in the blood. Which form is transported is controversial,

with interpretation supporting (i) an electroneutral net NH3

transport driven either by a chemical NH3 gradient, or by a

NHþ4 =Hþ exchange mechanism driven an electrochemical a

NHþ4 gradient coupled to a transmembrane proton gradient,

(ii) a direct NH3 transport associated with an induced NHþ4transport and (iii) an electrogenic transport of NHþ4 , partic-

ularly in plants [43,44]. Of note, the discovery that a

W148L substitution in AmtB of E. coli is associated with

higher activity and preferred transport of NHþ4 led some

investigators to conclude, contrary to their previous

reports, that AmtB, like plant Amts, might transport

charged substrates [45].

These different conclusions may reflect differences in the

expression systems used, notably those in which endoge-

nous ammonium transporter pathways are induced (Xeno-

pes). Nevertheless, Rh glycoproteins were the first

ammonium transporters identified in vertebrates. In

Fig. 1 Comparison of 3D structures of ammonium transporters (EcAmtB, NeRh50 and RhCG) with the predicted model of human RhAG. (Top): Amino acid

residues of the conduction pore viewed from a cross section cutting the pore of EcAmtB (pdb1u7g), NeRh50 (pdb 3b9w) and RhCG (pdb 3hd6) monomers

(left). The pore of human RhAG templated on the RhCG structure is shown on the right. Residues present in the external vestibule, the pore and the cyto-

plasmic vestibule and surface accessible to solvent are highlighted. The residues are shown in a Ca-only configuration of the main chain. (Bottom): Models

showing the ‘CO2 binding pocket’ of NeRh50 in which a CO2 molecule has been visualized, and the homologous region of human RhAG templated on RhCG

(pdb 3hd6). Because the space is occupied by residue Q273 (highlighted), a CO2 pocket in this position is unlikely in RhAG (Courtesy of Dr Isabelle Callebaut,

UMR7590, Université Pierre et Marie Curie, Paris).

30 J.-P. Cartron


mammals, they are present on RBCs, as well as in tissues

known to play a role in ammonium metabolism or excre-

tion such as kidney, liver and gastrointestinal tract of vari-

ous animals [42]. In RBCs, RhAG might play a role in the

transport of ammonium to detoxifying organs like the kid-

ney and the liver (but a role in the fine tuning of intracellu-

lar pH could also be evoked), whereas the non-erythroid Rh

glycoproteins might play a role in the regulation of the sys-

temic acid–base balance, at least in the kidney. Indeed,

RhCG is expressed in human distal nephron (with both api-

cal and basolateral localization) and exhibits increased

expression in response to chronic metabolic acidosis [46].

Although RhBG is present in mouse but not in human kid-

ney [47], expression studies in kidney cell lines (MDCK,

HEK293) have shown that basolateral targeting and ammo-

nium channel function of recombinant RhBG are regulated

by both phosphorylation and membrane skeleton binding

of the C-terminal cytoplasmic domain Ankyrine G [48,49].

Knockout of the Rhbg and Rhcg in mice described below

provide further insights into the role of these glycoproteins

in kidney.

The discovery that Rh-related glycoproteins may serve

as ammonium channels has been critical to understanding

the mechanism of ammonia excretion by aquatic animals.

In fish, ammonia is directly excreted into the environment

across the gill and to a lesser extent the skin. Multiple Rh

homologues (Rhbg, Rhcg2, Rhp2) are expressed in the gill,

including Rhag which is also present on fish RBCs. Based

on physiological studies in vivo and in vitro, it was pro-

posed that ammonia excretion might be coupled with a Na+

uptake process essential for living in freshwater conditions.

The model relies on a ‘Naþ=NHþ4 exchange complex’ con-

sisting of Rhcg (assumed to be a NH3 gas channel), vacuo-

lar-type H+-ATPase, Na+ ⁄ H+ exchanger and a epithelial

Na+ channel working together as a metabolon [reviewed in

50]. Some experimental support to this model was recently

provided by electrophysiological studies of MRC cells of

the skin from the Japanese fish medaka [51]. Of note, there

is no experimental evidence yet showing a physical cou-

pling between Rhcg1 and the Na+ ⁄ H+ exchanger.

Because the cellular systems used to explore the function

mediated by Rh-related proteins may contain potential

endogenous transporters and ⁄ or acid–base regulating pro-

teins that might interfere with ammonium transport, recon-

stitution in proteoliposomes was used to probe the

ammonium transport function of highly purified or recom-

binant AmtB protein from E. coli [25,52]. The method has

also been used recently to show that human recombinant

RhCG protein purified from HEK293 transfectant cells is a

fully functional NH3 transporter in the absence of any part-

ner and can be reversibly inhibited by mercuric and copper

salts [53]. Thus, reconstituted proteoliposomes should be

helpful to study the transport function and substrate

selectivity and specificity, as well as factors regulating

transport rate, of native or mutant Rh proteins, alone or in

association with other protein components.

CO2 transport

Although Rh glycoproteins clearly function as NH3 trans-

porters, a putative transport of CO2 is under debate [see 42

and references herein]. This is an attractive possibility

because the main function of RBCs is to mediate O2 ⁄ CO2

exchange in lung and tissues. Such function could be medi-

ated by a macrocomplex made of the physical association

between Band 3, the Rh complex, cytoskeletal and cytosolic

proteins assumed to form a metabolon [18]. One experi-

mental proof supporting this hypothesis in humans is based

on the determination of RBC membrane permeability to

CO2 (PCO2). The method uses a mass spectrometer to quan-

tify 18O-exchange between a solution of 18O-labelled

HCO�3 =CO2 and RBCs [54]. In this system, RBCs from com-

mon individuals (RhD-positive and RhD-negative) and

those from variants deficient for hUTB1 ⁄ Jk (urea trans-

porter), DARC ⁄ Fy, Kell and Kx proteins have a high PCO2

(�0Æ15 cm ⁄ s). By contrast, RBCs from Rhnull individuals

(lacking the Rh complex) and from COnull individuals (lack-

ing the water channel AQP1) have a severe reduction of

PCO2 (�0Æ07 cm ⁄ s), suggesting that the high CO2 permeabil-

ity of the human RBC membrane is equally caused by RhAG

and AQP1. The CO2 pathway constituted by Rh proteins

was inhibitable by NH4Cl and by DIDS, but not by DiBAC

(another inhibitor of anion exchange mediated by Band 3).

Thus, the Rh complex acts as a gas channel for CO2,

although this channel may not be selective for one gas, but

probably allows passage of NH3 as well as CO2 [54], and

perhaps O2 and NO [18]. As the lower PCO2 permeability of

Rhnull and AQP1null RBCs could not be detected by stopped-

flow analysis [55], double knockout Aqp1 ⁄ Rhag mice have

been generated [G. Gros, D. Goossens, JP Cartron et al.,

unpublished data], which provides an animal model to fur-

ther probe the gas permeability of RBCs.

Although N. europaea apparently shows no evidence of

CO2-dependent growth [28], it is interesting that CO2 pres-

surization experiments have revealed the presence in the

NeRh50 crystal of a ‘CO2 binding pocket’ in a deep cavity

near the pore exit [29]. However, in silico modelling sug-

gests that a CO2 pocket at this position is unlikely for

RhAG (Fig. 1). That RhAG and AQP1 greatly contribute to

the high permeability of the RBC membrane to CO2 can be

seen as an optimization of the physiological conditions of

CO2 exchange in lung and tissues [54]. Because AQP and

Rh family members seem permeable to CO2 and NH3, the

relative CO2 ⁄ NH3 selectivities of AQP1, AmtB and RhAG

have been compared by monitoring CO2- and NH3-induced

changes in surface pH of Xenopus laevis oocytes

Blood groups 31


expressing recombinant proteins [56]. The results indicate

that the three channels are permeable to CO2 and NH3, but

the relative index of permeability varies in the order

AQP1 > AmtB > RhAG. Thus, AQP1 is relatively more

selective for CO2 and AmtB ⁄ RhAG more selective for NH3.

Of note, the CO2 conductance of AQP1 and its biological

significance is controversial [57,58]. However, molecular

dynamics simulations have shown that aside from its role

in water conduction, the tetrameric AQP1 channel is

permeable to small neutral gas molecules and that a hydro-

phobic ‘central pore’ located at the centre of the four mono-

mers is predominantly permeable to CO2 and O2 whereas

the ‘monomeric water pore’ located at the centre of each

subunit may pass some CO2 but very few O2 [40].

Whether Rh protein homologues may play a role in CO2

flux across membranes in fish has been currently examined

only in rainbow trout, with a negative conclusion for Rhbg

and Rhcg mostly based on the absence of changes in

expression in gills and skin of animals under high CO2

exposure [59]. However, Rhag transcript levels responded

differently to high CO2 and high NH3 suggestive of a dual

role in NH3 and CO2 transport in trout RBCs.

Cation transport

Surprisingly, RBCs from individuals carrying heterozygous

mutations of RhAG (I61R and F65S) exhibit a severe mono-

valent cation leak (Na+, K+) [35], which is typically seen in

a rare form of haemolytic anaemia named overhydrated

hereditary stomatocytosis, OHSt [60,61]. This leak is the

residual cation permeability when the Na+K+ATPase and

the Na+K+2Cl) cotransporters are inhibited with ouabain

and bumetanide. Expression of the RhAG mutant proteins

in Xenopus laevis oocytes induces also a cation leak, but

surprisingly a weak cation leak (6 times lower) was found

with wild-type RhAG (but strict specificity was not demon-

strated). Structural modelling indicates that the F65S sub-

stitution is predicted to open the pore structure sufficiently

to allow the translocation of hydrated Na+, K+ and NHþ4ions (hydrated radii in the range of 3Æ31–3Æ58 A). The I61R

mutation opens the pore but to a lesser extent; additionally,

the two mutations might also cause long-range structural

alterations. These findings demonstrate that OHSt is caused

by RHAG gene mutations and suggest that RhAG might

mediate a basal cation flux. Whether these RhAG mutants

have retained a normal capacity to transport NH3 and ⁄ or

CO2 is unknown. Of note, in OHSt the mutated RhAG pro-

teins are expressed at the RBC surface (slight decrease),

whereas in Rhnull RBCs (regulator type), the mutated

proteins are absent. That a single-point mutation may alter

the substrate specificity of a transporter has precedents in

the literature. For instance, Band 3 mutations observed

in hereditary stomatocytosis convert the electroneutral

anion exchanger to a non-selective cation conductive

pathway [62].

In conclusion, the transport function of the Rh glycopro-

teins is still a matter of debate as there is no agreement on

the substrate(s) transported, either NH3=NHþ4 or CO2, and if

these proteins have the capacity to transport both sub-

strates, depending upon local physiological conditions and

of cells or tissues under examination (RBCs, kidney, etc.).

Differences between species also exist, as illustrated by

ammonium transport in plants, which is clearly electro-

genic, thus indicating a different underlying mechanism

for ionic substrate conduction [42–44]. A further complex-

ity is expected, as other gases such as O2 and NO might rep-

resent putative substrates [18,63]. The determination of 3D-

structure of Rh-related proteins is a crucial step for struc-

ture ⁄ function analysis and many approaches for studying

the mechanistics of the substrate translocation and pore

selectivity are under investigation, including molecular

dynamic simulations and functional reconstitution in pro-

teoliposomes with native and mutant proteins. Whether Rh

proteins function as macromolecular complexes in vivo in

association with other cell protein components to form me-

tabolons, as postulated in human RBCs and some tissues of

freshwater fishes, should be also carefully investigated as it

could reveal what could be the exact physiological role of

these proteins. There is some redundancy in NH3 and CO2

gas transport because other membrane proteins may also

be involved (aquaporins, urea transporters) and, in addition

to these complex factors, simple gas diffusion across cell

membranes may occur. Another challenge will be to deter-

mine the relative contribution of these mechanisms.

Fy ⁄DARC in health and disease

In addition to its central importance in transfusion medi-

cine, the Duffy (FY) blood group protein through its ‘recep-

tor function’ for plasmodial parasites, inflammatory

chemokines and HIV-1 virus is now considered an impor-

tant factor in normal physiology and the physiopathology

of various diseases.

Interaction with Plasmodium vivax

The Duffy (Fy) blood group protein is a well-known recep-

tor for P. vivax merozoites and for the related simian para-

site P. knowlesi.

Plasmodium vivax prevalence is very limited in West

Africa where about 95% Africans from these regions are

homozygous for a FY*Fy allele [causing the Fy(a-b-) phe-

notype] and resist invasion [64]. The promoter region of the

FY*Fy allele carries a mutation ()46C ⁄ C) in the binding site

of the GATA-1 transcription factor, preventing gene tran-

scription and Fy protein expression in erythroid tissues (not

32 J.-P. Cartron


in endothelial tissues), which might represent an adaptative

response to resist malaria [65]. The recognition site between

the malarial parasite and the Fy protein has been character-

ized to some extent, for both partners [66]. On the parasite

side, the Duffy recognition site was mapped to the con-

served cysteine-rich region II of PkDBL and PvDBL

(P. knowlesi and P. vivax Duffy binding-like) proteins that

bind to the extracellular N-terminus domain of the Fy pro-

tein, in which a sulphated tyrosine at position 41 plays a

central role (affinity increase up to 1000-fold). On the red

cell side, the parasite DBL domain binds to a 35 amino acid

region of N-terminal Fy protein that includes the Fy6 anti-

genic motif (centred on FE dipeptide at positions 22–23),

the Fya ⁄ Fyb polymorphic site (D42G protein substitution)

and the sulfated tyrosine-41. Interestingly, rabbit and

human antibodies against PvDBP region II reduce invasion

efficiency of P. vivax isolates in vitro by about 60% [67].

Moreover, naturally acquired antibodies to PvDBP region II

present in a cohort of children residing in an hyperendemic

area of Papua New Guinea confer partial protection against

P. vivax infection in vivo [68]. These results suggest that a

PvDBP-based vaccine might reduce human blood-stage P.

vivax infection. In that perspective, cocrystallization of the

parasite DBL domain with the N-terminus of the Fy protein

should define all contact residues involved in the binding

site and provide key information for the development of a

vaccinal strategies to block parasite entry in red cells [66].

However, genetic variation in P. vivax isolates may compli-

cate the development of this blood-stage vaccine [69]. Of

note, some strain variants of P. vivax may infect Fy(a-b-)

RBCs, suggesting still uncharacterized alternative path-

way(s) of invasion [70].

Interaction with chemokines

The Fy protein is also a promiscuous receptor which binds

with high affinity (Kd = 5 nM) to inflammatory chemokines

of the CXC and CC families such as CXCL1 (MGSA), CXCL8

(IL8), CCL2 (MCP-1) and CCL5 (RANTES) [for reviews see

71,72]. Accordingly, it was renamed DARC for ‘Duffy Anti-

gen ⁄ Receptor for Chemokines’. Of note, DARC binds to

CXC chemokines of the angiogenic type, which have an

ELR motif, but not to the angiostatic type. DARC belongs to

the G protein–coupled superfamily of receptors with

7-transmembrane domains that mediate leucocyte chemo-

taxis, but it is an ‘atypical receptor’ which behaves as a

‘chemokine-binding protein’ because it lacks the DRY motif

for trimeric G-protein coupling and does not transduce a

signal (by conventional pathways), although it can inter-

nalize ligands, but without their degradation, at variance

with the other atypical receptors D6 and CCX-CKR [73].

The seven transmembrane domains of DARC are

connected by four extracellular domains (including the

N-terminus of 63 residues which carries the Fya ⁄ b and Fy6

antigens as well as sulphated residues at positions 30 and

41 and all these domains in close association through

disulphide bonds participate in the chemokine-binding

pocket [74]. A structural protein-protein docking model

between DARC and CXCL8 has been recently proposed [75].

As many other chemokine receptors, DARC forms constitu-

tive dimers in living cells, either by homodimerization or

by heterodimerization, for instance with the HIV-1 core-

ceptor CCR5 (both are present on EC and use CCL5 ⁄ RANTES

as ligand), which results in down-regulation of the CCR5-

mediated responses and impaired chemotaxis [76].

DARC is not restricted to RBCs, but is also present at the

apical and basal plasma membrane (and caveolae) of endo-

thelial cells lining post-capillary venules, the primary site

of leucocyte transmigration from blood to organs, thus sug-

gesting a role of this protein in inflammatory reactions.

DARC is also present on Purkinje neurons in the cerebel-

lum, but the functional significance is unknown. On endo-

thelial cells lining post-capillary venules, DARC might play

a pro-inflammatory role by transporting chemokines pro-

duced in extravascular tissues across the endothelial barrier

(transcytosis), perhaps in cooperation with glycosamino-

glycans, and by presenting these chemokines to leucocytes,

thus promoting diapedesis and their recruitment to inflam-

mation sites [77]. On RBCs, DARC may play an anti-inflam-

matory role by serving as a ‘sink’ for chemokine removal

from sites of overproduction during inflammation, thereby

limiting leucocyte desensitization which impairs their emi-

gratory response [78]. Recent studies suggest that DARC

probably acts as a chemokine ‘buffer’ or ‘reservoir’, to regu-

late plasma chemokine levels [79]. In other words, DARC

binds chemokines locally where concentrations are high

and releases chemokines systemically when concentrations

fall below the Kd value [72].

Interestingly, the D42G polymorphism which determines

the Fya ⁄ Fyb blood group specificity is associated with 20%

variability in the serum concentration of the chemokines

CCL2 (MCP-1), CCL5 (RANTES) and CXCL8 (IL8). As this

polymorphism is not associated with difference in DARC

expression on RBCs, a structural ⁄ functional change in

DARC may be responsible for the difference in chemokine

binding [80]. Thus, the D42G polymorphism is a major reg-

ulator of DARC-mediated chemokine binding and of the

circulating concentration of several chemokines. The lack

of association of this polymorphism with CCL2 concentra-

tion in EDTA plasma led to the finding that blood clotting

(and heparin at high concentration) resulted in significant

release of CCL2 (also CCL5 and CXCL8) from DARC RBCs,

which may have clinical implications in the physiopathol-

ogy of cancer, inflammation and thrombosis.

As DARC is expressed on RBCs and specialized endothe-

lial cells, it plays a complex role in chemokine

Blood groups 33


homoeostasis by modulating the concentration of chemo-

kines in blood and by acting in endothelial transcytosis and

presentation of cytokines to leucocytes. Moreover, DARC

may compete for ligands shared with other chemokine

receptors and ⁄ or form heterodimers with chemokine recep-

tors, for instance with CCR5 on endothelial cells, which in

turn may negatively or positively alter the chemokine-

mediated signalling and cell response. These complex inter-

actions might explain discordant results on DARC implica-

tion in some pathological conditions, both in human

disease and murine models.

DARC-deficiency in man and mouse

In normal conditions, no obvious inflammatory disorders

were noticed in individuals lacking DARC either on RBCs

[Fy(a-b-) Blacks] or both on RBCs and endothelial cells,

suggesting some redundancy in DARC function. We already

mentioned that the Fy ⁄ DARC null status (DARC )46C ⁄ Cpromoter mutation) confers resistance to P. vivax infection.

Moreover, recent studies also conclusively established that

persons of African ancestry exhibit a benign ‘ethnic neutro-

penia’ which is associated with the DARC )46C ⁄ C genotype

[81].

To decipher the biological role of DARC on RBCs and

endothelial cells under pathological conditions, a number

of studies, sometimes contradictory, have been conducted

with either cohorts of patients including individuals of

African descent where about 70% lack DARC on RBCs

(DARC )46C ⁄ C promoter mutation) or animal studies

including wild-type and DARC-knockout mice as well as

DARC transgenic mice. Some of the results reported previ-

ously were based on such studies and others are summa-

rized in the following paragraphs.

Studies carried out with knockout mice that lack DARC

on endothelial cells but not on RBCs confirm that endothe-

lial DARC is an important molecule required for the proe-

migratory function of chemokines [82], which might

provide an explanation of why DARC is up-regulated in

inflammatory and infectious diseases (acute renal rejection,

crescentic glomerulonephritis, HIV nephropathy, HIV-asso-

ciated uremic syndrome, vasculitis, rheumatoid arthritis,

inflamed synovia, etc.), but the mechanism(s) involved is

(are) not known.

In African American men, there is a 60% higher inci-

dence of prostate cancer and a twofold higher mortality

than in Caucasian men. Although socio-economic factors

cannot be excluded, it seems that the absence of DARC on

RBCs might eliminate a mechanism by which angiogenic

chemokines are normally cleared from the tumour environ-

ment to restrict tumour progression [83]. Some support to

this view was provided in a transgenic model of prostate

cancer with DARC-deficient mice [84], although this should

be considered with caution because these animals lack

DARC both on RBCs and on endothelial cells.

DARC-deficient mice are fertile and survive normally.

However, the serum concentration of chemokine is higher

and there are abnormalities (leucocyte infiltration in lungs

and liver) in a model of endotoxemia. Recent clinical trials

of endotoxemia [Fy(a-b-) Blacks vs. Fy-positive Caucasians

receiving LPS intravenously] have shown that DARC sub-

stantially alters chemokine concentrations in blood

(CXCL1, CXCL8, CCL2), but does not have a protective

effect on the inflammation response (similar plasma levels

of TNFa and IL6) [85].

Overexpression of DARC may regulate growth and meta-

static potential of tumours (prostate, breast, lung, mela-

noma) in murine models, by clearing angiogenic CXC

chemokines from the tumour and inhibiting neovascular-

ization [86]. It was also reported that tumour cells dissemi-

nating from a primary tumour that expresses the

tetraspanin CD82 (synonym KAI1), a previously identified

suppressor of metastasis, may interact with endothelial

DARC, as revealed by a yeast two-hybrid assay, and that

this interaction ultimately leads to inhibition of cancer cell

proliferation at distant sites and to the senescence of

tumour cells [87]. These findings, however, await further

confirmation.

Preliminary evidence in mice suggests that DARC might

be one factor regulating bone mineral density (BMD), which

is determined by a balance between bone formation (by os-

teoblasts) and bone resorption (by osteoclasts). Osteoclast

formation is under the control of chemokines such as CCL2

and CCL5 that are ligands of DARC. Based on the findings

that BMD is increased in DARC-knockout mice and that

antibodies to DARC inhibits the formation of multinucleat-

ed osteoclasts in vitro, it was suggested that in normal ani-

mals DARC may negatively regulate BMD by increasing

osteoclast formation [88]. Of note, an association with bone

mineral density was not found in a large cohort of African

Americans with the DARC )46C ⁄ C promoter genotype [81].

Very recently, it was shown that the loss of Fy ⁄ DARC

antigen and of its chemokine scavenging function during

storage of RBCs amplifies lung inflammation in endotoxe-

mic mice. As reduction of Fy ⁄ DARC is also one conse-

quence of the storage lesion of human RBCs, these findings

suggest that transfusion of stored RBCs might augment

lung inflammation in critically ill patients [89].

DARC in HIV-1 susceptibility and disease progres-sion

Recent evidence suggests that HIV-1 virus particles bind to

RBCs via the Fy ⁄ DARC protein and it is anticipated that

erythrocyte-bound HIV-1 might represent a surface reser-

voir for trans-infection of permissive CD4+ cells. Moreover,

34 J.-P. Cartron


the DARC )46C ⁄ C promoter genotype, that abolishes ery-

throid expression of DARC on RBCs and confers P. vivax

resistance, might increase HIV-AIDS susceptibility in Afri-

can Americans, although it caused slower disease progres-

sion in HIV-1 infected patients [90]. One possible

explanation might be that the presence ⁄ absence of DARC

may influence plasma levels of HIV-1 suppressive and pro-

inflammatory chemokines. As the DARCnull phenotype is

by itself highly informative about ancestry in African

American populations some controversy about these find-

ings has been raised, based on a possible inadequate correc-

tion for population stratification. Independent studies have

also conclusively established that persons of African ances-

try exhibit a ‘benign ethnic neutropenia’ which is associ-

ated with the DARC )46C ⁄ C genotype [81]. As leukopenia

is frequently observed during HIV-1 infection and is associ-

ated with an accelerated HIV-1 disease course, it was shown

that this effect was more prominent in leukopenic subjects

of European than African ancestry and that leukopenic but

not non-leukopenic HIV(+) African Americans with DARC

)46C ⁄ C genotype had a survival advantage compared with

all Fy-positive subjects [91]. The mechanism(s) involved

are not clear but suggest that a complex interaction

between DARC genotype, chemokines and leukopenia

might be associated with a survival advantage in HIV-

infected African Americans. Of note, the blood group Pk

antigen (globotriaosylceramide, Gb3) also strongly influ-

ences HIV infection, as high Pk expression correlated with

low HIV infection [92].

Further studies on the implication of DARC in various

cellular functions should provide invaluable information

on the pathogenesis of various diseases or disorders such as

malaria, inflammation reactions, cancer metastasis, osteo-

porosis and HIV infection.

Genetic disorders affecting blood groups andtheir impact on function

Investigations of rare ‘null phenotype’ variants that are

defective for blood group antigens provide insights into the

cell membrane organization and the function of blood

group antigens. Additionally, as some blood groups exhibit

a broad tissue distribution, clinical or subclinical manifes-

tations may extend to non-erythroid tissues. The manage-

ment of null phenotypes is crucial because in all instances

they confer a high risk of immunization by transfusion or

pregnancy.

Null phenotypes with a mild or severe syndrome

Several null phenotypes are associated with mild-to-mod-

erate haemolytic conditions, indicating some role for blood

group molecules in red cell membrane integrity or function.

This was illustrated by three phenotypes associated with

the defect of Rh-RhAG complex (Rhnull syndrome), Kx-Kell

complex (McLeod syndrome) and glycophorins C and D

(Leach phenotype) which have been extensively investi-

gated and has revealed that these molecules are present in

the membrane as molecular complexes [for reviews see

11,15,93–99]. Defects of Band 3 can also be considered here

as this protein, which is the major red cell membrane com-

ponent with multiple functions (anion exchanger, mem-

brane skeleton linkage, P. falciparum invasion and

cytoadhesion, red cell senescence, membrane macrocom-

plex ⁄ gas metabolon) [reviews 18,100–106], is now recog-

nized as the carrier of the blood group DI (Diego) antigen.

Band 3 alterations (deficiency, mutations) in RBCs and ⁄ or

in kidney (where a N-ter truncated form is present) have

clinical implications in various disorders such as hereditary

spherocytosis, hereditary stomatocytosis, distal renal tubu-

lar acidosis (dTRA) (with or without RBC alterations) and

South Asian Ovalocytosis (SAO) [for reviews see

61,102,106–110].

In contrast to examples mentioned earlier, a number of

null phenotypes caused by the absence or severe lack of

blood group proteins with a well defined function are clini-

cally silent, at least in basal conditions. Typical examples

include COnull, JKnull, Kellnull (K0), LUnull (recessive), LWnull

or Fynull. Either the function of these proteins is not vital or

there may be functional redundancy. Alternatively, the

phenotype might only be revealed under certain stress or

pathological conditions. For instance, under water depriva-

tion, AQP-1 deficient individuals exhibit a defective uri-

nary concentrating ability and a decreased pulmonary

vascular permeability [111]. Similarly, sickle red cell adhe-

sion to vascular endothelium is increased via an erythroid

pathway implicating the PKA-dependent phosphorylation

of LU and ICAM-4 (LW) glycoproteins and binding to endo-

thelial ligands (laminin for LU, aVb3 integrin for ICAM-4),

which in turn may contribute to vaso-occlusive episodes in

sickle cell disease [112–114] and such adhesive effects may

be modulated upon treatment [114]. Another example,

described earlier, is the role of Fy ⁄ DARC protein in patho-

gen interactions and inflammatory diseases.

Phenotypes associated with altered function of theerythroid KLF1 transcription factor

A rare mild red cell disorder caused by the severe lack of

red cell antigen Lutheran (Lu) occurs with a red cell

restricted suppression of antigens such as CD44, CD151,

AnWj (erythroid Haemophilus influenza receptor) and the

P1 glycolipid. It is caused by a gene ‘inhibitor of Lutheran’

unlinked to the LU locus, called In(Lu), which is dominantly

inherited. The molecular basis of the In(Lu) phenotype

found in 21 of 24 individuals is related to various

Blood groups 35


mutations, at the heterozygous state, in the promoter or

coding sequence of EKLF ⁄ KLF1 (Erythroid Kruppel-like

Factor), a transcription factor involved in erythroid differ-

entiation [115]. In some individuals, the In(Lu) phenotype

is characterized by an abnormal red cell morphology (mild

poikilocytosis, acanthocytosis), but there is no anaemia

[116], thus indicating that a single functional KLF1 gene is

sufficient for normal erythropoiesis. The osmotic fragility is

normal, but during incubation (24 h at 37�C) the cells lose

K+ and become osmotically resistant. Of note, Lunull pheno-

types caused either by homozygosity for a very rare reces-

sive lu gene at the LU locus (chromosome 19), or by

hemizygosity for an uncharacterized X-linked suppressor

gene, exhibit a normal red cell morphology and no abnor-

mal electrolyte transport. Various mutations (nonsense,

deletion) of the lu gene causing the recessive type of Lu(a-

b-) phenotype have been identified [117], but the molecular

basis of the X-linked phenotype is still unknown.

Interestingly, in a new form of dyserythropoietic anae-

mia described in a patient presenting a persistence of

embryonic and foetal haemoglobins, there is an erythroid-

restricted defect characterized by the lack of CD44 (carrier

of Indian antigens) and of the water channel AQP-1 (carrier

of Colton antigens). This patient, therefore, has the extre-

mely rare phenotype In(a-b-), Co(a-b-) [118], and recent

investigations have detected a mutation (E325K) of the

KLF1 protein [119]. Another patient with a similar, but not

identical, form of dyserythropoietic anaemia showing a

complete defect of CD44, AQP1 and ICAM-4 with the

E325K mutation was independently identified and charac-

terized [Arnaud et al., manuscript in preparation]. This is

consistent with the critical role of KLF1 in the regulation of

many target genes [120], suggesting that the blood group

defects in these patients are most likely secondary but that

the gene(s) causative of the dyserythropoiesis is (are) not

identified yet.

A CD44 defect also occurs in 4Æ1-deficient RBCs. Addi-

tionally, there is a sharp decrease in CD47, suggesting that

both proteins participate in the 4Æ1R-based multiprotein

junctional complex (4Æ1 ⁄ p55 ⁄ GPC) critical for the mechani-

cal properties of the red cell membrane [121]. Moreover, a

Ca2+-dependent modulation of CD44-protein 4Æ1-AnkyrinR

interaction has been described before [122]. Of note, CD47

is also involved in the Band 3 macrocomplex linked to the

membrane skeleton via AnkyrinR.

CD151 and kidney disease

Recently, CD151 (carrier of MER2 antigens; RAPH blood

group system) has been identified on human red cells.

CD151 is a member of the tetraspanin superfamily of pro-

teins which facilitates the interaction of membrane and

intercellular signalling molecules by formation of specific

microdomains [123]. Three patients of Indian Jewish origin

with end-stage kidney disease were found to be homozy-

gous for a single-nucleotide insertion (G383) in exon 5 of

the CD151 gene, causing a frameshift and premature stop

signal in codon 140 [124]. The CD151 defect nephropathy

is associated with pretibial epidermolysis bullosa and deaf-

ness, suggesting that CD151 may be essential for the correct

assembly of basement membranes in the human kidney

and may have functional significance in the skin and the

inner ear. The patients have severe anaemia attributable, at

least in part, to the coexistence of b-thalassaemia minor,

but there is an impaired marrow response to erythropoietin.

Murine models of CD151 defect have also been obtained.

Insights into blood group gene function andphysiology from gene targeting

Gene targeting by homologous recombination in embry-

onic stem cells to create a null mutation (‘knockout’, KO) in

mice (or other vertebrate ⁄ invertebrate species) is a powerful

technique for clarifying the biological activities of genes

and to develop physiological investigations that cannot be

performed in humans. In recent years, gene ‘knockdown’

by RNA interference, a mechanism for silencing gene

expression by targeted degradation of mRNA, was also

developed as a further experimental tool for deciphering

the function of biomolecules and is a promising approach

for designing new therapeutic strategies [125].

Several genes encoding blood group proteins have been

disrupted in mice (Table 1). Some of these results have been

discussed in several reviews [97–99], including the

FY ⁄ DARC KO discussed earlier.

Regarding RBC function, the most significant finding of

targeting studies was the severe red cell phenotype

observed in mice targeted for erythroid Band 3 [126] or for

both the erythroid and kidney isoforms [127]. Briefly, the

animals were severely anaemic and there was a high mor-

tality at birth and growth retardation. Accelerated erythro-

poiesis reflected by an increased reticulocytosis and a

marked hepatomegaly ⁄ splenomegaly was noted. Red cells

were strikingly spherocytic with a pronounced loss of sur-

face area, as seen in human hereditary spherocytosis, but

the cells assemble an architecturally nearly normal mem-

brane skeleton. Protein 4Æ2 and GPA were absent, support-

ing the critical role of band 3 for protein 4Æ2 attachment

and as a chaperone for the transport of GPA to the cell sur-

face [128,129]. These findings are consistent with severe

Band 3 deficiencies observed in humans [61]. Of note, Band

3 deficiency resulting from a severe phenotype caused by a

spontaneous mutation in mice (wan ⁄ wan) [130] and in cat-

tle [131] have also been reported.

In contrast to Band 3, targeting of blood group-related

genes in other published examples did not compromise

36 J.-P. Cartron


Table 1 Blood group gene invalidation in mice by homologous recombination

Targeted gene

Phenotype of ‘knockout (KO)’ mice

RBC phenotype Other features

BAND 3 (CD233) Severe haemolytic anaemia; Spherocytosis (shading of

membrane vesicles); Absence of 4Æ2 protein and GPA; reduced

level of ankyrin, Rh, Rhag. CD47 level normal; Nearly

normal skeletal architecture

Fertile. High mortality in prenatal period; Growth retardation;

Mild effect on acid–base balance regulation in kidney

(More severe phenotype in the wan ⁄ wan mouse mutant

lacking Band 3)

GPA (CD235A) TER antigen absent; Decreased electric charge; Slight increase

in osmotic fragility after 24 h at 37�C; Normal Band 3 level

Fertile. Normal development

CD44 RBC function not impaired Fertile. Defective progenitor egress from bone marrow;

No critical role in erythroid differentiation; Exaggerated

response to infection (Cryotosporidium parvum); No

developmental or neurologic defects. Role in tumour growth

DAF (CD55) No increase in spontaneous complement activation in vivo.

Increased bystander complement activation and enhanced

complement deposition in vitro, but no haemolysis


CD47 RBC function not impaired. Marker of ‘self’ on RBCs. CD47 on

RBCs interacts with SIRPa on macrophages to inhibit RBC

clearance

Fertile. Normal development; Decreased resistance to bacterial

infection. Defect in granulocyte function; Decreased cytokine-

stimulated osteoclast formation; CD47 is a regulator of bone

mass; Lack of CD47 on non-hematopoietic cells induces

macrophage tolerance to CD47null cells; Vascular cells from

CD47null mice do not respond to TSP-1-induced NO-mediated

vascular relaxation. CD47 is the critical TSP-1 receptor

regulating vascular response to NO. CD47 targeting increases

survival of ischaemic tissue. CD47null phenotype not described

in humans

CD147 RBC function not impaired Sterile. Role in early embryogenesis and reproduction; No

abnormality of blood brain barrier; Increased mitogenic

response in MLR; Retinal dysfunction and degeneration of

photoreceptor cell function

DARC (CD234) No apparent phenotype. No binding of chemokines (CC ⁄ CXC)

Mature RBCs, not reticulocytes, resist invasion by P. yoelii

Fertile. Normal development; Abnormal leucocyte mobilization

in model of endotoxemia. Negative regulation of bone mineral

density?

ICAM-4 (CD242) No apparent phenotype Fertile. Impaired erythroblastic island formation

LU-BCAM (CD239) Lu absent from WT mouse RBC. No apparent phenotype Fertile. Normal development; Abnormal basement membrane in

glomerular kidney and smooth muscle intestine; No renal

dysfunction under basal conditions

CD151 No apparent phenotype Fertile. Renal failure (glomerulosclerosis) on FVB background,

but no phenotype on C57Bl ⁄ 6 background

AQP1 No apparent phenotype. Reduced osmotic water permeability Fertile. Slight growth retardation; Urine concentrating

capability impaired

AQP3 AQP3 absent from WT mouse RBC. No apparent phenotype.

Reduced glycerol permeability

Fertile. Normal development; Urine concentrating defect

and skin defects

UT-B No apparent phenotype. Reduced urea permeability Fertile. Normal development; Severe urinary concentration

defect; Early maturation of reproductive system in male;

Cardiac conduction defect

RH (CD240) No apparent phenotype. Normal haematological parameters;

No ICAM-4; Rhag reduced by 30% only; CD47 normal;

Slight reduction of NH3 permeability


RHag (CD241) No apparent phenotype. Normal haematological parameters;

No ICAM-4; no Rh; CD47 normal; Severe reduction of NH3

permeability


RHbg Absent from WT human and mouse RBC Fertile. Normal development; No role in renal distal tubular

acidosis

Blood groups 37


RBC survival or function, although, specific functions such

as water or urea permeabilities, or parasite infection for

instance were severely reduced or abolished in some

instances (JK ⁄ UT-Bnull, AQP1null, Fy ⁄ DARCnull) with no

clinical consequences, at least under basal conditions.

However, some dysfunctions in non-erythroid tissues,

sometimes severe, were occasionally observed [97–99]

(Table 1).

A notable finding deduced from the CD47 KO led to the

unexpected discovery that this molecule is a ‘marker of

self’, at least in mice. Although CD47 was not reported to

carry any blood group antigen, it is associated with the Rh

complex on human mature RBCs, and major findings

regarding this interesting molecule will be summarized in

the following sections. This will be followed by a brief sum-

mary of KO published within the last few years such as

those of Cd151, Lu ⁄ bcam, Icam-4(LW), Rh, Rhag (and ana-

logues Rhbg ⁄ Rhcg), and Kel.

CD47 in mice and humans

CD47 is a ubiquitous membrane protein of the Ig superfam-

ily which interacts with integrins and its cellular ligands

are thrombospondin-1 (TSP-1) and immune inhibitory

receptors of the signal regulatory protein (SIRP) family

[132,133]. CD47 is also present on human mature RBCs

which normally do not bear detectable integrins. CD47 is

involved in multiple functions from immune homoeostasis,

including phagocytosis, apoptosis, cell migration or T cell

responses, to the regulation of neuronal networks [134].

CD47 KO mice exhibit characteristics summarized in

Table 1. Studies based on these mice have shown that CD47

on RBCs and other cells is a regulator of phagocytosis

through interaction with the inhibitory receptor SIRPa on

macrophages. CD47 was therefore considered as a ‘marker

of self’ that protects autologous cells from removal by mac-

rophages [135]. The CD47-SIRPa interaction may also limit

the destruction of host cells sensitized by antibody or com-

plement in animal models of autoimmune diseases [136].

Recently, studies using bone marrow chimeras have shown

that the lack of CD47 on non-hematopoietic cells induces

macrophage tolerance to CD47null RBCs by an unknown

mechanism [137], thus explaining why CD47) ⁄ ) RBCs sur-

vive in KO mice. Interestingly also, the better engraftment

of human haematopoietic stem cells achieved in the NOD-

SCID mice model of xenotransplantation could be

explained by a polymorphism in SIRPa, as the variant car-

ried by the macrophages from this strain of mice showed

enhanced binding to human CD47 compared to those from

other immunodeficient mouse strains [138]. CD47 is tran-

siently upregulated on cytokine-mobilized hematopoietic

stem cells in the blood to avoid phagocytosis and CD47

expression is constitutively elevated on human and mouse

leukaemic progenitors as an important mechanism by

which they could escape macrophage killing, thus promot-

ing their enhanced survival and engraftment [139]. Thus,

CD47 might be a new target for therapeutic intervention in

leukaemia. Further studies with CD47 KO mice revealed

other interesting functions of CD47. First, through interac-

tion with its receptor TSP-1, which is known to limit the

angiogenic and vaso dilatator activities of NO, CD47 inhib-

its NO signalling. Therefore, strategies that block the TSP-

1 ⁄ CD47 interaction greatly enhances ischaemic tissue sur-

vival in animal models of ischaemia–reperfusion by

improving vascular relaxation and blood flow, which might

have promising therapeutic applications [140,141]. Second,

based on the observation that CD47 KO mice have an

increased bone mass and defective osteoclast function in

vivo, it was shown that CD47 can regulate osteoclastogene-

sis and bone mass through modulation of NO production,

and interaction with the avb3 integrin, two factors known

to play a role in developing osteoclasts in vitro [142].

Whether or not CD47 is a marker of self in humans and

might play similar functions as those described earlier in

mice is not so clear-cut. In humans, it was suggested that

the mild haemolytic anaemia associated with the Rhnull

syndrome might be secondary to a sharp reduction (80–

90%) of CD47 on Rhnull RBCs [135]. However, Rhnull RBCs

interact like control RBCs with peripheral macrophages ex

vivo in the monocyte monolayer assay and are not phago-

cytosed to a higher extent [143]; in the mouse study, how-

ever, CD47-deficient RBCs were removed by splenic

resident macrophages, not bone marrow-derived macro-

phages [123]. RBCs from D.., D– and RN donors have a

Table 1 (Continued)

Targeted gene

Phenotype of ‘knockout (KO)’ mice

RBC phenotype Other features

RHcg Absent from WT human and mouse RBC Distal renal tubular acidosis; Rhcg required for epididymal fluid

pH homoeostasis and male fertility

KEL (CD238) Reduced level of XK; No ET-3 converting enzyme activity;

increased Gardos channel activity

Fertile. Normal development; Mild alteration of some motor

activities suspected?

38 J.-P. Cartron


moderate reduction (20–50%) of CD47 but there is no sign

of haemolysis [17]. Further, human 4Æ2) ⁄ ) RBCs, but not

4Æ2 KO mouse RBCs, have a strong reduction (80%) of CD47

[17] and similarly, some human 4Æ1) ⁄ ) RBCs [121], but not

4Æ1 KO mouse RBCs [144], are also defective in CD47, thus

indicating that the lack or severe reduction of CD47 is not

responsible for the anaemia associated with 4Æ2 or 4Æ1 defi-

ciency [17]. These findings indicate that murine and human

RBCs differ in the expression, membrane organization and

cell-type specificity of CD47-SIRPa interactions [145].

Using human monocytes ⁄ macrophages as effector cells

and opsonized RBCs (sheep and human) or recombinant

human CD47 coated to microbeads as targets, Tsai and Di-

scher [146] have shown that signalling through CD47-

SIRPa interaction occurs with formation of a ‘phagocytic

synapse’. Moreover, they found that about 10–20% of the

normal CD47 density of normal RBCs is sufficient to deliver

a signal inhibiting phagocytosis. In the absence of a human

CD47null phenotype, these findings might explain that RBCs

with reduced levels of CD47 from most human variants

described earlier can be tolerated. Accordingly, CD47-

SIRPa signalling should not be the primary mechanism,

when compared to PS exposure for instance, by which

stored or senescent human RBCs are removed from the cir-

culation because those cells lose only a limited fraction

(about 10–50%) of CD47. Although CD47-deficient NOD-

SCID mice develop a severe autoimmune haemolytic anae-

mia (AIHA) [136], CD47 levels on RBC and SIRPa levels on

monocytes ⁄ macrophages were similar between human

patients with AIHA and ⁄ or immune thrombocytopenia and

healthy controls [147,148]. These findings underline the

complexity of red cell destruction mechanisms and the deli-

cate balance between prophagocytic (Fc receptors, comple-

ment regulatory proteins) and antiphagocytic (SIRP) signals

[149,150].

Cd151 gene targeting

Mice with targeted disruption of the Cd151 gene, which

encodes a tetraspanin carrier of MER2 antigens (RAPH sys-

tem) in humans, are grossly normal and healthy on a

C57Bl ⁄ 6 background [151], but Cd151 KO on a mixed

FVB ⁄ N ·129 background results in a severe glomerular dis-

ease [152], as observed in humans [124]. Most interestingly,

however, Cd151 KO mice on C57Bl ⁄ 6 background develop

a severe glomerular disease associated with proteinuria

after backcross on a FVB background [153] and further

studies suggested that Cd151, which is present in podocytes

on the mouse kidney, could be involved in assembly and

maturation of the glomerular basement membrane in col-

laboration with integrins a3b1 [153]. Therefore, Cd151

appears as a critical adhesion molecule involved in the

podocyte-glomerular basement membrane interaction.

These findings point to the critical importance of mouse

genetic background in such studies and to the potential role

of gene modifier(s) that may influence the onset of a

disease.

Lu ⁄ bcam gene targeting

Mice with targeted disruption of the Lu ⁄ bcam gene, which

encodes the Lu protein, a receptor of laminin 511 ⁄ 521 in

humans, are viable, fertile and develop normally [154].

Thus, Lu ⁄ bcam KO did not reproduce the severe defects seen

in mice lacking laminin ?5 (which die during late embryo-

genesis). However, Lu ⁄ bcam KO animals exhibited struc-

tural alterations of basement membranes expressing

laminin-511 ⁄ 521 in kidney and intestine, two organs

known to highly express Lu ⁄ bcam. The loss of Lu ⁄ bcam

was associated with a thickened basement membrane per-

turbing the organization of intestinal smooth muscle layers

and of the glomerular basement membrane without any

apparent functional defect in basal conditions [154]. Of

note, like KO mice, rare individuals that lack all Lutheran

blood group antigens (natural Lunull of the recessive type)

exhibit no phenotype and no clinical syndrome under

physiological conditions, but for obvious reasons, this

could not be explored further.

Icam-4 gene targeting

Mice with targeted disruption of the Icam-4 gene, which

encodes the LW protein, an adhesion molecule with a broad

substrate specificity (b2- and aV-integrins) in humans, are

viable and fertile, but exhibited a defect in the formation of

‘erythroblastic islands’ (bone marrow niche composed of a

central macrophage surrounded by developing erythro-

blasts) both in vivo and in reconstitution assays in vitro,

although haematocrit, haemoglobin and red cell indices

were normal [155]. Thus, in basal conditions, Icam-4 KO

mice have no anaemia or any red cell phenotype, but stress

hematopoiesis was not investigated. In humans, individuals

with the rare LWnull phenotype (ICAM-4 deficiency) are

apparently healthy and their RBCs express the Rh proteins

normally.

Rh and Rh-related genes targeting

Mice invalidated for the erythroid-specific genes Rh and

Rhag have been reported recently [39]. These mice were not

anaemic and showed no obvious abnormalities in erythro-

cyte parameters, blood cell count, blood cell morphology

and fragility as observed in the Rhnull syndrome in humans

[15]. Histology of spleen and bone marrow was normal.

However, expected biochemical alterations in RBCs were

present. RBCs from Rhag KO animals lacked Rh, Rhag and

Blood groups 39


Icam-4 proteins and those from Rh KO animals lacked Rh

and Icam-4, but Rhag was reduced by 30% only. RBC levels

of CD47 were not affected in both types of mice. Rhag KO

RBCs exhibited a severe defect in NH3 transport, whereas

Rh KO RBCs, which retained substantial amounts of Rhag

proteins had a moderate reduction of transport. Rh KO and

Rhag KO RBCs also exhibited a decreased basal adhesion to

an endothelial cell line most likely resulting from defective

Icam-4 membrane expression. These findings underline dif-

ferences between the human and murine models of Rh defi-

ciency and the structure of the Rh complex in the two

species. Whether or not the lack of phenotype in these mice

might be related to their genetic background is unknown.

Mice invalidated for both genes recapitulated the defects

found in each single KO but they showed no difference in

recovery from phenylhydrazine-induced haematopoietic

stress when compared to wild-type animals, suggesting that

the lack of Icam-4 did not affect stress erythropoiesis [39],

as might be expected from Icam-4 KO [155].

Of note, mice deficient for the non-erythroid homo-

logues Rhbg and Rhcg have been generated recently

[156,157]. Both Rhbg and Rhcg proteins belong to the

Amt ⁄ Mep family of ammonium transporters and were

expressed in tissues involved in ammonium metabolism

(kidney, liver, etc.) [24]. Gene targeting of Rhbg did not

alter renal acid handling or hepatic ammonium metabo-

lism, and therefore the biological function of this protein

remains unclear [156]. Although RhBG deficiency has not

been reported in humans, no incidence is expected in

human kidney as RhBG is not expressed in this tissue [47].

Mice lacking Rhcg had abnormal urinary acidification

because of impaired ammonium excretion on acid loading,

a feature of distal renal tubular acidosis [157]. Moreover,

the Rhcg protein was found present in epididymal epithelial

cells and required for epididymal fluid pH homoeostasis

and normal male fertility. RhCG is expressed in human kid-

ney [46,47], but to date no RhCGnull phenotype has been

reported. Mice with a collecting duct-specific Rhcg deletion

have also been generated [158]. Under basal conditions,

urinary NH3 excretion was decreased in these mice com-

pared to controls, but urine pH was unchanged. After acid-

loading for 7 days, the KO mice developed a more severe

metabolic acidosis than did control mice. Thus, Rhcg

expression contributes to both basal and acidosis-stimu-

lated renal ammonia excretion. Whether or not RhCG dis-

plays similar functions in humans is unknown because

RhCG-deficiency has not been reported in man yet.

Other studies have shown that knockout of the Rh50-like

homologous gene in Dictyostelium discoidum has no appar-

ent effect [159], whereas knockdown of Rhcg1 in zebrafish

embryos decreases ammonium excretion [160] and knock-

down of CeRh1 in the nematode Caenorhabtitis elegans is

lethal despite the presence of endogenous Amt genes [161].

Kel gene targeting

In mice with targeted disruption of the kel gene, RBCs

lacked Kell glycoprotein, Kell-Xk complex, endothelin-3

enzyme converting activity, and have a reduced level of Xk

protein [162]. Interestingly, the Gardos channel activity

(Ca2+-activated K+ channel, KCNN4) was increased, but the

normal enhancement by ET-3 was blunted. Other defects

have been reported such as a reduced intratumoural vascu-

larization of induced Lewis lung carcinoma and possibly a

mild motor dysfunction, but these latter effects should be

confirmed on a greater number of animals.

Targeting of blood group genes in mice is a useful

approach to better understand the function of the genes

products in normal or pathological conditions in humans.

However, these studies also revealed significant differences

between man and mouse. For example, contrary to human,

murine RBCs lack Lu ⁄ BCAM, AQP3, Glut1 and red cell

CD47 expression is not affected in Rh ⁄ Rhag, 4Æ1 and 4Æ2 KO

mice. Similarly, some blood group changes (Rh, Kell, Fy)

revealed by investigations of 4Æ1 KO mice [144] are not seen

in 4Æ1-deficient human RBCs [121]. Further, Rhbg is present

in mouse but not human kidney and other differences are

likely to exist. Such differences should be taken into

account in our models to understand red cell structure and

cell physiology.

Conclusion

Through considerable progress made in the biochemistry,

genetics and molecular biology, the function and biological

role of a growing number of blood group molecules is now

emerging.

All these studies have illustrated the large diversity of

functional activities reported, and the potential physiologi-

cal roles of blood group and blood group-related molecules

cover large areas of human physiology from cerebral to

renal and reproduction biology. As specific deficiencies of

these molecules have only a minor or no detrimental effect

in most instances, they are either dispensable for cell func-

tion or redundant. In some instances, however, a function

becomes apparent under stress or in pathological condi-

tions. Alternatively, such molecules may represent evolu-

tionary vestiges devoid of a significant function. How

common and rare blood group polymorphisms are main-

tained and may impact function is largely unknown,

although a few examples clearly point to selective pressure

exerted by pathogenic micro-organisms.

Despite significant progress, much remains to be discov-

ered to clearly delineate how blood group molecules, alone

or as molecular complexes in erythroid and non-erythroid

cells, may act in health and disease, to understand the

underlying mechanisms, and, ultimately, how these

40 J.-P. Cartron


findings might eventually be translated into clinical

applications.

Acknowledgements

I wish to thank Dr Dominique Goossens (INTS, Paris) for

critical reading and English reading of the manuscript and

Dr Isabelle Callebaut (CNRS, UMR7590, Universite Pierre et

Marie Curie Paris 6, Paris) for the structural models of Rh

proteins.

Disclosures

The author declares no conflicts of interest.

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Blood groups 45


blood groups: genetics and physiology

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